Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus which anchors them to the membranes of organelles where they mediate critical cellular processes. Accordingly, mutations in genes encoding TA proteins have been identified in a number of severe inherited disorders. Despite the importance of correctly targeting a TA protein to its appropriate membrane, the mechanisms and signals involved are not fully understood. In this study we identify additional peroxisomal TA proteins, discover more proteins which are shared by multiple organelles and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelle locations in mammals. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We show that subtle changes in those parameters can shift TA proteins between organelles, explaining why peroxisomes and mitochondria share many TA proteins. This enabled us to associate characteristic physicochemical parameters with particular organelle groups. Using this classification allowed successful prediction of the location of uncharacterized TA proteins for the first time.